Acetaminophen (p-hydroxyacetanilide) can result in acute renal damage in man when ingested in large amounts. Acetaminophen and its sulfate and glucuronide conjugates are also the major metabolites in man of phenacetin (p-ethoxyacetanilide), a compound implicated in the pathogenesis of analgesic nephropathy. Because our previous studies demonstrated that acetaminophen can be metabolized by the liver to a toxic intermediate species, we have investigated the possibility that extra-hepatic activation by the kidney might occur in a similar manner. Acetaminophen given subcutaneously was found to produce acute tubular necrosis in Fischer rats and to covalently bind to renal and hepatic tissue macromolecules. Pretreatment with inhibitors of the P-450 system decreased covalent binding and protected against histologic tissue damage. Acetaminophen was converted by rat kidney microsomes to reactive metabolites that became covalently bound to microsomal protein. The covalent binding required NADPH and was inhibited by carbon monoxide. These experiments suggests that acetaminophen overdoses in man occurs by a similar mechanism. Studies are now in progress to determine the significance of these observations to the pathogenesis of analgesic nephropathy.